Adenylyl cyclase from S. cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which has now been called cyclase-associated protein (CAP). A cDNA encoding CAP has been cloned by screening a yeast cDNA expression library in E. coli with antisera raised against the purified protein. The cDNA contains an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases. Adenylyl cyclase activity in membranes from cells that lack CAP is not stimulated by RAS2 proteins in vitro. These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system. Mutants lacking CAP have four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology (Field, 1990).
SRV2 has been identified as a gene that alleviates stress sensitivity in yeast strains carrying an activated RAS gene. Epistasis analysis suggests that the gene affects accumulation of cAMP in the cell. Direct assays of cAMP accumulation indicate that mutations of the gene diminish the rate of in vivo production of cAMP following stimulation by an activated RAS allele. Null mutations of srv2 result in lethality, which cannot be suppressed by mutational activation of the cAMP-dependent protein kinase. The sequence of the gene indicates that it encodes an adenylate cyclase-associated protein. These results demonstrate that SRV2 protein is required for RAS-activated adenylate cyclase activity, but that it participates in other essential cellular functions as well (Fedor-Chaiken, 1990).
A new 56 kDa actin-binding protein (ASP-56) was isolated from pig platelet lysate. In falling ball viscosimetry it causes a reduction in viscosity that can be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD-labelled actin shows reduction of polymeric actin, too. These results can be explained by sequestering of actin in a non-polymerizable 1:1 ASP-56/actin complex. Sequencing of about 20 tryptic peptides of ASP-56 and comparison with known sequences reveals about 60% homology to the adenylate cyclase-associated protein (CAP) from yeast (Gieselmann, 1992).
The adenylyl cyclases of both S. cerevisiae and S. pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, degenerate oligonucleotides were designed and used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, a human cDNA clone encoding a 475-amino-acid protein was isolated that is homologous to the yeast CAP proteins. Expression of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals (Matviw, 1992).
CAP, a protein from S. cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins. CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess. A molecular and phenotypic analysis of the CAP protein has been carried out. The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes. Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways (Gerst, 1991).
cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast S. pombe, has been identified, cloned, and studied. CAP protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast S. cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also map the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation are observed when the entire gene is disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. These results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP (Kawamukai, 1992).
By performing yeast two-hybrid screens, peptides have been identified from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. These observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. Human CAP has been shown to interact with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. Actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts (Hubberstey, 1996).
cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2, have been amplified and cloned. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap minus strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap minus strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap minus cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins (Yu, 1994).
The carboxyl terminus of yeast CAP has been shown to sequester actin, but whether this function has been conserved, and is the sole function of this domain, is unclear. The carboxyl-terminal domains of CAP and CAP homologs have been shown to have two separate functions. Carboxyl-terminals of both yeast CAP and a mammalian CAP homolog, MCH1, bind to actin. This domain contains a signal for dimerization, allowing both CAP and MCH1 to form homodimers and heterodimers. The properties of actin binding and dimerization are mediated by separate regions on the carboxyl terminus; the last 27 amino acids of CAP being critical for actin binding. Evidence is presented that links a segment of the proline-rich region of CAP to its localization in yeast. Together, these results suggest that all three domains of CAP proteins are functional (Zelicof, 1996).
The role of the actin cytoskeleton in Ras/cAMP signaling has been analyzed. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduce adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also fails to localize but shows no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduce CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A does not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling (Yu, 1999).
CAP is a component of the S. cerevisiae adenylyl cyclase complex. The N-terminal domain is required for cellular RAS responsiveness. Loss of the C-terminal domain is associated with morphological and nutritional defects. cap minus cells bud randomly and are defective in actin distribution. The morphological and nutritional defects associated with loss of the CAP C-terminal domain are suppressed by over-expression of PFY, the gene encoding profilin, an actin- and polyphosphoinositide-binding protein. The phenotype of cells lacking PFY resembles that of cells lacking the CAP C-terminal domain. Study of mutated yeast profilins and profilins from Acanthamoeba suggests that the ability of profilin to suppress cap- cells is dependent upon a property other than, or in addition to, CAP's ability to bind actin. This property may be its ability to bind polyphosphoinositides. It is proposed that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton (Vojtek, 1991).
The S. cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. Evidence shows that CAP sequesters monomeric actin (Kd in the range of 0.5-5 microM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots have identified the 46-kDa protein as yeast actin. CAP also binds to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrate that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers (Freeman, 1995).
In search for novel actin binding proteins in Dictyostelium discoideum, a cDNA clone coding for a protein of approximately 50 kDa has been isolated that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in E. coli and used in actin binding assays. CAP sequesters actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain lead to an up to 50% fluorescence enhancement of pyrene-labeled G-actin, indicating a direct interaction, whereas the amino-terminal domain does not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation is approximately two times higher than that of intact CAP. The sequestering activity of full length CAP can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone is not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells, CAP is enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein is very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner (Gottwald, 1996).
The CAP (cyclase-associated protein) homolog of D. discoideum is a phosphatidylinositol 4,5-bisphosphate [PIP(2)] regulated G-actin sequestering protein that is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. GFP-fusions of N- and C-domains were constructed and it was found that the N-terminal domain shows CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They have an altered cell morphology with very heterogeneous cell sizes and exhibit a cytokinesis defect. Growth on bacteria is normal both in suspension and on agar plates, as is phagocytosis of yeast and bacteria. In suspension in axenic medium, mutant cells grow more slowly and do not reach the saturation densities observed for wild-type cells. This is paralleled by a reduction in fluid phase endocytosis. Development is delayed by several hours under all conditions assayed, furthermore, motile behaviour is affected (Noegel, 1999).
Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). Experiments were designed to address CAP1 regulation of the actin cytoskeleton. CAP1 localizes to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, GFP-CAP1 fusion proteins were constructed and it was found that fusion proteins lacking the actin-binding region localize like the wild type protein. Microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts have found that antibodies attenuate serum stimulation of stress fibers. CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulate the formation of stress fiber-like filaments when microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton (Freeman, 2000).
S. cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region. SH3 (Src homology 3) domains have been shown to bind to proline-rich regions of proteins. The proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. The SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p have been found to complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein that complexes with adenylyl cyclase. CAP homologs from S. pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin (Freeman, 1996).
CAP is a multifunctional protein; the N-terminal region binds adenylyl cyclase and controls its response to Ras while the C-terminal region is involved in cytoskeletal regulation. In between the two regions, CAP possesses two proline-rich segments, P1 and P2, resembling a consensus sequence for binding SH3 domains. Two yeast proteins have been identified with molecular sizes of 48 and 46 kDa associated specifically with P2. Determination of partial protein sequences demonstrated that the 48-kDa and 46-kDa proteins correspond to EF1 alpha and rL3, respectively, neither of which contains any SH3-domain-like sequence. Deletion of P2 from CAP results in loss of the activity to bind the two proteins either in vivo or in vitro. Yeast cells whose chromosomal CAP has been replaced by the P2-deletion mutant display an abnormal phenotype represented by dissociated localizations of CAP and F-actin, which are colocalized in wild-type cells. These results suggest that these associations may have functional significance (Yanagihara, 1997).
Cofilin-ADF (actin-depolymerizing factor: Drosophila homolog Twinstar) is an essential driver of actin-based motility. Two proteins, p65 and p55, were discovered that are components of the actin-cofilin complex in a human HEK293 cell extract and p55 was identified as CAP1/ASP56, a human homologue of yeast CAP/SRV2 (cyclase-associated protein). CAP is a bifunctional protein with an N-terminal domain that binds to Ras-responsive adenylyl cyclase and a C-terminal domain that inhibits actin polymerization. Surprisingly, the N-terminal domain of CAP1, but not the C-terminal domain, is responsible for the interaction with the actin-cofilin complex. The N-terminal domain of CAP1 accelerates the depolymerization of F-actin at the pointed end; depolymerization is further enhanced in the presence of cofilin and/or the C-terminal domain of CAP1. Moreover, CAP1 and its C-terminal domain facilitate filament elongation at the barbed end and stimulate ADP-ATP exchange on G-actin, a process that regenerates easily polymerizable G-actin. Although cofilin inhibits the nucleotide exchange on G-actin even in the presence of the C-terminal domain of CAP1, its N-terminal domain relieved this inhibition. Thus, CAP1 plays a key role in speeding up the turnover of actin filaments by effectively recycling cofilin and actin and through its effect on both ends of actin filament (Moriyama, 2002).
Dynamic remodeling of the actin cytoskeleton requires rapid turnover of actin filaments, which is regulated in part by the actin filament severing/depolymerization factor cofilin/ADF. Two factors that cooperate with cofilin are Srv2/CAP and Aip1. Human CAP enhances cofilin-mediated actin turnover in vitro, but its biophysical properties have not been defined, and there has been no in vivo evidence reported for its role in turnover. Xenopus Aip1 forms a cofilin-dependent cap at filament barbed ends. It has been unclear how these diverse activities are coordinated in vivo. Purified native yeast Srv2/CAP forms a high molecular weight structure comprised solely of actin and Srv2. The complex is linked to actin filaments via the SH3 domain of Abp1. Srv2 complex catalytically accelerates cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhances the ability of profilin to stimulate nucleotide exchange on ADP-actin. Yeast Aip1 forms a cofilin-dependent filament barbed end cap, disrupted by the cof1-19 mutant. Genetic analyses show that specific combinations of activities mediated by cofilin, Srv2, Aip1, and capping protein are required in vivo. It is concluded that two genetically and biochemically separable functions have been defined for cofilin in actin turnover. One is formation of an Aip1-cofilin cap at filament barbed ends. The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex. The Srv2 complex is shown to be a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization (Balcer, 2003).
Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, gaining an understanding ofthe mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells has demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects as those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals (Bertling, 2004).
Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, evolutionarily conserved residues have been described that mediate binding to ADP-G-actin; these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, these data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly (Mattila, 2004).
In hydra, head activator (HA) acts as positive signal for nerve-cell determination and differentiation. For both events, HA uses cAMP as the second messenger. Evidence is presented that the cAMP agonist, Sp-cAMPS, is able to mimic the effect of HA on nerve-cell determination and differentiation and that it is blocked by the antagonist Rp-cAMP. An adenylyl cyclase associated protein, CAP, appears to be involved as mediator for transducing the signal from the transmembrane HA receptor to the cAMP system. A cDNA coding for hydra CAP was isolated from the multiheaded mutant of Chlorohydra viridissima. The hydra CAP shows extensive homology with the yeast and, more so, mammalian CAPs. In hydra, CAP mRNA is expressed abundantly in interstitial and epithelial cells. The effect of HA, but not of cAMP, on nerve-cell differentiation is inhibited by pretreatment of hydra with a cap antisense oligonucleotide, suggesting a role for CAP as mediator in the signal transduction cascade between HA and cAMP (Fenger, 1994).
Actin-depolymerizing factor (ADF)/cofilins (see Drosophila Twinstar) contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins (see Drosophila Capulet) block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. By monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, this study directly quantify the reactions underpinning filament severing and depolymerization from both ends. In the absence of monomeric actin, soluble ADF/cofilin was found to associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. It was also shown that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. These experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, the results propose that significant barbed-end depolymerization may take place in cells (Wioland, 2017).
A living cell's ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin (see Drosophila Twinstar) and filament cappers like capping protein (CP; see Drosophila Capulet) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF), filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, this study provides the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. Using a conceptually novel assay to directly visualize ADF's effect on a population of pre-assembled filaments, it was demonstrated how ADF's enhanced pointed-end depolymerization causes an increase in polymerizable actin monomers, thus promoting faster barbed-end growth. It was further reveale that ADF-enhanced depolymerization synergizes with CP's long-predicted "monomer funneling" and leads to skyrocketing of filament growth rates, close to estimated lamellipodial rates. The "funneling model" hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few non-capped filaments transiently grow much faster, an effect proposed to be very important for motility. This study provides the first direct microscopic evidence of monomer funneling at the scale of individual filaments. These results significantly enhance understanding of the turnover of cellular actin networks (Shekhar, 2017).
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